The genome organization of the sequenced 4.6-Mb human Major Histocompatibility Complex (MHC) will be best understood in a comparative evolutionary context. To develop a physical map of the domestic cat MHC for nucleotide sequencing, we have constructed two large insert domestic cat genomic DNA libraries, one using the P1 artificial chromosome (PAC) system with an average insert size of 80 kb and a second using the bacterial artificial chromosome (BAC) system with an average insert size of 137 kb which has been arrayed in 384-well plates. State-of-the-art, high-throughput analysis technologies are being applied to BAC/PAC contig mapping of the feline MHC. Using 46 human and/or feline MHC DNA probes, we identified 490 feline BAC and 66 feline PAC DNA clones containing sequences which hybridize to MHC probes. Putative MHC positive BAC/PAC clones were arranged in a 96-well plate format and (a) rescreened by colony hybridization and (b) BAC/PAC DNAs were purified for fingerprint analysis and Southern hybridization. To generate contig maps of large insert clones, complete Hind III-digested BAC/PAC DNAs were fingerprinted by electrophoresis and the data analyzed using the Fingerprinting Contigs (FPC) software developed by C. Soderlund which assembles a minimal tiling path of clones using an algorithm to cluster clones into contigs based on their probability of coincidence scores. Using these approaches, we have constructed a sequence-ready 3.1 Mb BAC/PAC contig map of the domestic cat MHC consisting of 177 BAC and 18 PAC clones. The order of the five subregions of the human and mouse MHC named for their respective gene families: 1) extended class II; 2) classical class II; 3) class III; 4) classical class I; and 5) extended class I is conserved in the domestic cat. The feline MHC extended and classical class II region BAC/PAC contig map consists of 28 BACs and 12 PACs spans ~800 kbp. The gene order is similar to, but distinct from human and mice: ARE1, DPB/DPA, RING3, DMB, TAP1, DOB, DRB2, DRA3, DRB2, DRA2 and DRA1. However, apparently unlike the human and mouse MHC, both the domestic cat DRA and DRB genes have undergone multiple duplications, and the entire DQB/DQA region was deleted at some point in the evolutionary history of the domestic cat. The feline MHC class III region BAC/PAC contig map, consisting of 40 BACs and 17 ordered markers, spans ~600 kbp. The 17 mapped genes in this region spanning NOTCH4 to LTA is syntenic with both the human and mouse. The domestic cat classical and extended MHC class I region BAC/PAC contig map, consisting of 109 BAC and 6 PAC clones and 33 ordered markers, spans ~1.6-Mb. The data indicate that the feline classical and extended class I region spanning MIC-B to OLFR is syntenic to both human and mouse, but the region corresponding to the human HLA class I-B and HLA-C region has undergone considerable expansion during evolutionary history and contains at least 12 class I genes similar to the mouse H2-Q region. However, the region corresponding to HLA-E contains a single gene/gene-fragment unlike human and mouse and the HLA-A region has been deleted. Based on fluorescent in situ hybridization (FISH) analyses of selected feline class I and II BAC/PAC clones, the class II region maps to cat chromosome B2q11.1 and the class I region lies in the pericentromeric region of B2.